Duplex molecular assay intended for point-of-care diagnosis of influenza A/B virus infection.

Early diagnosis and management of influenza virus infection directly correlates with the effectiveness in disease control. Current molecular influenza virus tests were designed for use in diagnostic testing facilities, where sophisticated equipment and highly trained technicians are available. A longer turnaround time for the centralized testing than when testing near the sample source could delay the initiation of medical intervention, thereby reducing the efficacy of antiviral treatment. The new assay, the SAMBA (simple amplification-based assay) Flu duplex test, is a dipstick-based molecular assay developed to provide a simple, accurate, and cost-effective solution for the diagnosis of influenza A/B viruses intended for near-patient testing. The test presents an alternative format of influenza virus molecular testing that utilizes isothermal amplification and visual detection of nucleic acid on a test strip. The entire test procedure (extraction, amplification, and detection) is integrated into an enclosed semiautomated system. Analytically, the SAMBA Flu duplex test detects 95 and 85 copies of viral genomes for influenza A and B viruses, respectively, with no cross-reactivity observed against other common respiratory pathogens. The clinical performance was established by blind testing of 328 nasal/throat and nasopharyngeal swab specimens from the United Kingdom and Belgium and comparing the results with the quantitative reverse transcription-PCR method routinely used in two public health laboratories. The SAMBA Flu duplex test showed a clinical sensitivity and specificity of 100% and 97.9% for influenza virus A and 100% and 100% for influenza virus B. The test provides a new technology that could facilitate simple and timely identification of influenza virus infection, potentially resulting in more efficient control measures.

Simultaneous molecular detection and confirmation of influenza AH5, with internal control.

Influenza viruses continue to be a major cause of respiratory tract infection, resulting in substantial morbidity and mortality throughout the world. Accurate and rapid differential diagnosis of influenza virus infections, particularly associated with zoonotic infections, is important for public health actions, patient management, and treatment. Real-time PCR is widely considered the gold standard for molecular detection of influenza viruses owing to its high assay specificity, extreme detection sensitivity, and wide linear dynamic range. This protocol describes the use of a real-time RT-PCR assay for identification of influenza A and B viruses, detection of H5 subtype viruses, and an internal control, in a multiplexed, single-tube format. The inclusion of an internal bacteriophage control allows the efficiency of the extraction and amplification process to be monitored, so that false-negative results may be avoided. The primers and probe sets in this multiplex assay have been validated with a panel of influenza A viruses of different subtypes (including swine influenza viruses), and influenza B viruses, and specificity further confirmed with non-related respiratory viruses.

Nucleic acid dipstick test for molecular diagnosis of pandemic H1N1.

A new nucleic acid amplification-based rapid test for diagnosis of pandemic influenza (H1N1) 2009 virus was developed. The molecular test for pandemic H1N1, SAMBA (simple amplification-based assay), is based on isothermal amplification and visual detection on a dipstick characterized by high sensitivity, high specificity, a short turnaround time, and minimal technical requirements. The amplification step is monitored with an internal control to ensure correct interpretation of test results. The clinical performance of this assay was evaluated using blinded RNA samples extracted from nasal/throat swab specimens from 262 patients exhibiting influenza-like illness. Compared with the United Kingdom National Standard Method, based on quantitative reverse transcription-PCR, the sensitivity, specificity, positive predictive value, and negative predictive value of the new assay were 95.3% (95% confidence interval, 88.5 to 98.7%), 99.4% (95% confidence interval, 96.9 to 99.9%), 98.8% (95% confidence interval, 93.5 to 99.9%), and 97.8% (95% confidence interval, 94.4 to 99.4%), respectively. The SAMBA for pandemic H1N1 provides a new technology that could potentially facilitate timely diagnosis and management of infected individuals, thereby informing decision making with regard to patient isolation during a pandemic outbreak.

Establishment of a UK National Influenza H5 Laboratory Network.

Avian (H5N1) influenza continues to pose a significant threat to human health, although it remains a zoonotic infection. Sensitive and robust surveillance measures are required to detect any evidence that the virus has acquired the ability to transmit between humans and emerge as the next pandemic strain. An integral part of the pandemic planning response in the UK was the creation in 2005 of the UK National H5 Laboratory Network, capable of rapidly and accurately identifying potential human H5N1 infections in all regions of the UK, and the Republic of Ireland. This review details the challenges that designing molecular detection methods for a rapidly evolving virus present, and the strategic decisions and choices required to ensure successful establishment of a functional national laboratory network, providing round the clock testing for H5N1. Laboratory partnerships have delivered improved real-time one-step multiplex PCR methodologies to ensure streamlined testing capable of not only detecting H5 but also a differential diagnosis of seasonal influenza A/B. A range of fully validated real-time PCR H5 confirmatory assays have been developed to run in parallel with a universal first-screening assay. Regular proficiency panels together with weekly surveillance runs, intermittent on-call testing for suspect cases of avian flu in returning travellers, and several outbreaks of avian influenza outbreaks in poultry that have occurred since 2005 in the UK have fully tested the network and the current diagnostic strategies for avian influenza. The network has clearly demonstrated its capability of delivering a confirmed H5N1 diagnosis within 3-4 h of receipt of a sample, an essential prerequisite for administration of the appropriate antiviral therapy, effective clinical management, disease containment and implementation of infection control measures. A functional network is an important means of enhancing laboratory capability and building diagnostic capacity for a newly emerging pandemic of influenza, and is an essential part of pandemic preparedness.

Transmission of influenza A in families.

We conducted a prospective, nested study using nucleotide sequencing to examine influenza positive respiratory samples in families for genetic homology in the hemagglutinin and neuraminidase genes. Influenza A H3N2 viruses from 3 families had identical, family specific, HA1 nucleotide sequences. Sequences among these families were genetically heterogeneous. A 4th family was distinguished by sequencing of the influenza neuraminidase gene.

Design and validation of an H5 TaqMan real-time one-step reverse transcription-PCR and confirmatory assays for diagnosis and verification of influenza A virus H5 infections in humans.

Increasing diversity among influenza H5N1 viruses has resulted in the need for sensitive and specific diagnostic assays, fully validated for the detection of H5 viruses belonging to all hemagglutinin (HA) clades, particularly the recently circulating H5N1 viruses of clade 2. In this report, the development and validation of a real-time, one-step TaqMan reverse transcription-PCR (RT-PCR) assay specific for the detection of influenza A H5 viruses from clades 1, 1', 2, and 3 is described. The real-time assay for H5 virus was shown to be highly sensitive, detecting H5 virus levels of <1 PFU from each of the HA clades. Specificity of the H5 RT-PCR for influenza A H5 viruses was demonstrated by using influenza A viruses of different subtypes, clinical samples containing influenza A viruses H1N1, H3N2, and H5N1, influenza B viruses, and other respiratory viruses. The usefulness of the inclusion of a distinguishable assay positive control and of confirmatory assays for the laboratory diagnosis and verification of H5 virus infections was demonstrated. A real-time RT-PCR pyrosequencing assay, a restriction enzyme digestion assay, and direct sequencing of the H5 real-time RT-PCR amplicon were validated for the confirmation of H5 detection by the diagnostic real-time assay. The H5 real-time assay was applied to diagnostic testing for suspected cases of influenza A virus H5 infection in the United Kingdom. Influenza A H5 viruses were not detected in the cases analyzed; however, influenza A H3N2 virus was detected in 57% of the suspected cases of H5. The H5 TaqMan real-time RT-PCR and confirmatory assays will be useful tools for the laboratory surveillance and rapid diagnosis of H5 infections in humans.

Influenza AH1N2 viruses, United Kingdom, 2001-02 influenza season.

During the winter of 2001-02, influenza AH1N2 viruses were detected for the first time in humans in the U.K. The H1N2 viruses co-circulated with H3N2 viruses and a very small number of H1N1 viruses and were isolated in the community and hospitalized patients, predominantly from children <15 years of age. Characterization of H1N2 viruses indicated that they were antigenically and genetically homogeneous, deriving the hemagglutinin (HA) gene from recently circulating A/New Caledonia/20/99-like H1N1 viruses, whereas the other seven genes originated from recently circulating H3N2 viruses. Retrospective reverse transcription-polymerase chain reaction analysis of influenza A H1 viruses isolated in the U.K. during the previous winter identified a single H1N2 virus, isolated in March 2001, indicating that H1N2 viruses did not widely circulate in the U.K. before September 2001. The reassortment event is estimated to have occurred between 1999 and early 2001, and the emergence of H1N2 viruses in humans reinforces the need for frequent surveillance of circulating viruses.

Molecular diagnosis of influenza.

The past decade has seen tremendous developments in molecular diagnostic techniques. In particular, the development of PCR technology has enabled rapid and sensitive viral diagnostic tests to influence patient management. Molecular methods used directly on clinical material have an important role to play in the diagnosis and surveillance of influenza viruses. Molecular diagnostic tests that allow timely and accurate detection of influenza are already implemented in many laboratories. The combination of automated purification of nucleic acids with real-time PCR should enable even more rapid identification of viral pathogens such as influenza viruses in clinical material. The recent development of DNA microarrays to identify either multiple gene targets from a single pathogen, or multiple pathogens in a single sample has the capacity to transform influenza diagnosis. While molecular methods will not replace cell culture for the provision of virus isolates for antigenic characterisation, they remain invaluable in assisting our understanding of the epidemiology of influenza viruses.